Calcineurin is a Ca.sup.2 + /calmodulin-dependent protein phosphatase and is an element of many intracellular signaling pathways. Guerini and Klee, Proc. Natl. Acad. Sci. USA 86:9183-9187 (1989). The protein has been identified in eukaryotic cells ranging from yeast to mammals. Cyert and Thorner, J. Cell. Biol., 107:841a (1989) and Klee et al., Adv. Enzymol., 61:149-200 (1984). Because calcineurin may affect many signaling pathways in the same cell, some means of specific targeting of calcineurin's activity is required. One cellular means for specifically targeting enzyme activity in a cell is by compartmentalization. Compartmentalization provides a means for segregation of signaling pathways and contributes to the specificity of cellular responses to different stimuli. Compartmentalization of certain enzymes occurs by interaction of the enzymes with specific anchoring proteins. For example, cAMP-dependent protein kinase (PKA) is anchored at specific intracellular sites by binding to A-Kinase Anchor Proteins (AKAPs). Hirsch et al., J. Biol. Chem., 267:2131-2134 (1992). cAMP activates PKA by binding to the regulatory subunits (R) of the dormant PKA holoenzyme and causes the release of the active catalytic subunit (C). Two classes of R subunit exist; RI and RII which form the type I and type II PKA holoenzymes, respectively. The subcellular distributions of these PKA isoforms appear to be distinct. The RI isoforms (RI.alpha. and RI.sctn.) are reported to be predominantly cytoplasmic and are excluded from the nuclear compartment, whereas up to 75% of the RII isoforms (RII.alpha. or RII.sctn.) are particulate and associated with either the plasma membrane, cytoskeletal components, secretory granules, the golgi apparatus, centrosomes or possibly nuclei.
AKAPs have been identified in a variety of organisms. At least 7 proteins that bind the regulatory subunit of PKA in Aplysia californica, a marine invertebrate have been identified. Cheley et al., J. Biol. Chem., 269:2911-2920 (1994). One of these proteins is enriched in crude membrane fractions and taxol-stabilized microtubules and may thus anchor microtubules to the cell membrane as well as bind PICA. A mammalian AKAP has been identified that is related to microtubules; microtubule-associated protein 2 (MAP2) attaches PKA to the cytoskeleton. Threurkauf and Vallee, J. Biol. Chem., 257:3284-3290 (1982) and DeCamilli et al., J. Cell Biol., 103:189-203 (1986). The PKA-binding site on MAP2 is a 31-residue peptide in the amino-terminal region of the molecule. Rubino et al., Neuron, 3:631-638 (1989) and Obar et al., Neuron, 3:639-645 (1989).
Another AKAP that associates with microtubules, AKAP 150, accumulates in dendrites in close association with microtubules. Glantz et al., Mol. Biol. Cell, 3:1215-1228 (1992). AKAP 150 is present in several neuronal cell types and is a member of a family of AKAPs that are the principal AKAPs from mammalian brains. Other members of this family include AKAP 75 found in bovine brain and AKAP 79 found in human brain. Glantz et al., J. Biol. Chem., 268:12796-12804 (1993). AKAP 75 apparently binds cytoskeletal elements through two non-contiguous regions near the N-terminus of AKAP 75. AKAP 79 is predominantly present in postsynaptic densities (PSDs) in the human forebrain. Carr et al., J. Biol. Chem., 267:16816-16823 (1992). Inhibition of binding between AKAP 79 and PKA in hippocampal neurons has been shown to inhibit alpha-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid/kainate glutamate receptors. Rosenmund et al., Nature, 368:853-856 (1994).
Other AKAPs have also been characterized. Exposure of granulosa cells to follicle-stimulating hormone and estradiol has been demonstrated to up-regulate expression of an 80 kDa AKAP. Carr et al., J. Biol. Chem., 268:20729-20732 (1993). Another AKAP, Ht31, has been cloned from a human thyroid cDNA library. Carr et al., J. Biol. Chem., 267:13376-13382 (1992). Another AKAP, AKAP 95, changes its intracellular location during the cell cycle. AKAP 95 is an integral nuclear protein during interphase, but becomes associated with cytoplasmic PKA when the nuclear membrane breaks down during mitosis. This suggests that AKAP 95 could play a role in targeting activity of certain isoforms of PKA during cAMP-responsive events linked to the cell cycle. Coghlan et al., J. Biol. Chem., 269:7658-7665 (1994). Other known AKAPs include an 85 kDa AKAP which links PKA to the Golgi apparatus (Rios et al., EMBO J., 11:1723-1731 (1992)) and a 350 kDa AKAP that binds PKA to centromeres (Keryer et al., Exp. Cell Res., 204:230-240 (1993)).
The known AKAPs share a common PKA-binding feature. Although the primary structure of the AKAPs is not conserved, each has a secondary structure motif that includes an amphipathic helix region. Scott and McCartney, Mol. Endo., 8:5-11 (1994). Binding of AKAPs to the regulatory subunit of PKA is blocked by a peptide that mimics this helical structure of the PKA binding region of AKAPs. Disruption of the peptide's helical structure by an amino acid substitution abolishes the PKA-AKAP binding block (Carr et al., J. Biol. Chem., 266:14188-14192 (1991)), demonstrating that PKA binding occurs in the amphipathic helix of AKAPs and is governed by the secondary structure of the AKAP molecules. This provides a means for segregation of a kinase that, like calcineurin, is common to many signaling pathways yet may act in a pathway-specific manner.
One specific action of calcineurin is participation in T cell activation. Clipstone and Crabtree, Nature, 357:695-697 (1992) and O'Keefe et al., Nature, 357:692-694 (1992). In T cells, calcineurin participates in regulation of IL-2 expression following T cell stimulation. Weiss and Littman, Cell, 76:263-274 (1994). Nuclear factor of activated T cells (NFAT.sub.p) has been shown to be a substrate for calcineurin phosphatase activity. It has been suggested that, following T cell stimulation, calcineurin-mediated NFAT.sub.p dephosphorylation allows translocation of NFAT.sub.p from the cytoplasm to the nucleus where NFAT.sub.p interacts with Fos and Jun to induce expression of the IL-2 gene. Jain et al., Nature, 365:352-355 (1993).
Calcineurin's role in T cell activation provides a target for therapeutic intervention into T cell-mediated disorders and medications have been developed that inhibit calcineurin. Two calcineurin-inhibiting drugs, cyclosporin A (cyclosporin) and FK506, have been used in the clinic. Thomson and Starzl, Immunol. Rev., 136:71-98 (1993). Both cyclosporin and FK506 inhibit calcineurin only after binding to distinct intracellular proteins known as immunophilins (cyclophilin and FKBP 12, respectively). Schreiber and Crabtree, Immunology Today, 13:136-142 (1992). Thus, cyclosporin and FK506 act as prodrugs. Following binding to their respective immunophilins, the drug/immunophilin complexes bind calcineurin, thereby inhibiting the phosphatase activity.
Calcineurin inhibition has been most effectively exploited in the treatment of graft rejection following organ transplant. Cyclosporin and FK506 have been employed following renal, hepatic, cardiac, lung, and bone marrow transplants. The Canadian Multicentre Transplant Study Group, N. Engl. J. Med., 314:1219-1225 (1986); Oyer et al., Transplant Proc., 15:Suppl 1:2546-2552 (1983); Starzl et al., N. Engl. J. Med., 305:266-269 (1981); The Toronto Lung Transplant Group, JAMA, 259:2258-2262 (1988); and Deeg et al., Blood, 65:1325-1334 (1985). The use of these medications has significantly prolonged graft survival and lessened morbidity following transplant. Najarian et al., Ann. Surg., 201:142-157 (1985) and Showstack et al., N. Engl. J. Med., 321:1086-1092 (1989).
Cyclosporin also has been used in a variety of autoimmune-related diseases. Uveitis generally improves within a few weeks of therapy, but quickly relapses after cyclosporin is discontinued. Nussenblatt et al., Am J. Ophthalmol., 96:275-282 (1983). Similarly, psoriasis generally improves with cyclosporin therapy, but quickly relapses after treatment. Ellis et al., JAMA, 256:3110-3116 (1986). "Honeymoon" periods of insulin independence may be induced and prolonged in both new onset Type I and Type II diabetes mellitus when cyclosporin is administered within two months of insulin therapy. Feutren et al., Lancet, 2:119-124 (1986) and Bougneres et al., N. Engl. J. Med., 318:663-670 (1988). A variety of nephropathies, including minimal-change focal and segmental, membranous, and IgA-mediated nephropathies, may also be sensitive to cyclosporin, although observed reductions in proteinuria may be due to a decrease in the glomerular filtration rate and not healing of the basement membrane. Tejani et al., Kidney Intl., 29:206 (1986). Cyclosporin administration also has a dose-dependent effect on rheumatoid arthritis, although such treatment is associated with a high incidence of nephrotoxicity. F.o slashed.rre et al., Arthritis Rheum., 30:88-92 (1987).
As mentioned above, cyclosporin has been associated with nephrotoxicity. Mason, Pharmacol. Rev., 42:423-434 (1989). Depressed renal function occurs in virtually all patients treated with cyclosporin. Kahan, N. Engl. J. Med., 321:1725-1738 (1989). This can generally be reversed by cessation of cyclosporin therapy. Unfortunately, in organ graft recipients substitution of other commonly used immunosuppressives for cyclosporin carries a high risk of graft rejection. In renal transplant patients this can require reinstitution of dialysis. In patients that have received hearts, lungs, or livers, graft rejection can be fatal. Although less common than nephrotoxicity, neurotoxicity and hepatotoxicity are also associated with cyclosporin therapy. de Groen et al., N. Engl. J. Med., 317:861-866 (1987) and Kahan et al., Transplantation, 43:197-204 (1987).
Although there is less clinical experience, significant toxicity has also become apparent in the use of FK506. Like cyclosporin, FK506 is associated with significant nephrotoxicity. Peters et al., Drugs, 4:746-794 (1993). The clinical presentation, lesion morphology, and incidence are approximately equivalent to those of cyclosporin. McCauley, Curr. Op. Nephrol. Hyperten., 2:662-669 (1993). Neurotoxicity has also been associated with FK506. Eidelman et al., Transplant. Proc., 23:3175-3178 (1991) and Fung et al., Transplant. Proc., 23:3105-3108 (1991). In contrast to cyclosporin, FK506 has a hepatotrophic, rather than hepatotoxic, effect. Peters et al., supra.
In view of the significant potential toxicity of immunosuppressive agents, such as cyclosporin and FK506, it is clear that there is a need in the art for additional agents that inhibit calcineurin. These agents would preferably be associated with fewer toxic side effects than presently available agents and thus could provide an advance in immunosuppressive therapy.